The process of alternative splicing (AS) is a powerful mechanism for generating mRNA diversity from protein coding genes. Alternative mRNA isoforms from the same gene can differ subtly from each other or have radical alterations in their sequence composition. Remarkably, very little is known about whether or not this diverse pool of mRNA is converted to protein. One intriguing clue for solving this problem comes from our previous studies suggesting that the process of alternative splicing influences the translational efficiency of the resultant mRNA isoforms. In this proposal, we use both genome-wide and molecular approaches to untangle the intricate mechanisms coupling post-transcriptional gene expression. To accomplish this goal we propose the following specific aims: (1) Determine the prevalence and impact of AS-TC in human gene regulation. In this aim we will quantify mRNA isoforms with respect to the translating polyribosomes in 15 different ENCODE cell lines and two different primate stem cell model systems using our recently developed Frac-Seq methodology. These experiments will reveal the scope of AS-TC regulatory events and determine if this is a conserved mechanism for spatial or temporal regulation of gene expression. (2) Using the Serine and Arginine-rich protein SRSF1 as a model, we will elucidate the roles and targets of shuttling RNA binding proteins in coupling of post- transcriptional gene regulation. In this aim we will map the binding sites of SRSF1 or non-shuttling mutants in different subcellular fractions using CLIP-Seq; Determine the dependency of AS-TC on SRSF1 using Frac-Seq; Discover novel trans-acting AS-TC factors using RNA affinity chromatography. The results of this work will define a new paradigm for the function(s) of shuttling proteins such as SRSF1 in the coordination of post- transcriptional gene expression. (3) Determine the molecular mechanisms regulating isoform-specific mRNA translation. In this aim we will elucidate the full repertoire of RNA elements and structures that regulate polyribosome association using FragSeq and QUEPASA then determine biochemically how sequence elements involved in AS-TC modulate mechanisms of translation initiation and elongation. By determining how AS-TC elements regulate mRNA translation we will be able to refute or support the central hypothesis of this proposal, that alternative splicing generates isoforms with different translational efficiency. If sequences associated with differential polyribosome association do not directly control translation initiation or elongation, then we will con- sider our alternative hypothesis: that a significant fraction of mRNA isoform diversity arises from noisy splicing which are then excluded from polyribosomes by some unknown mRNA surveillance pathway. Solving this important problem will not only reveal how cis-elements influence translational yield, but will also define mechanistic links between the processes of alternative splicing and mRNA translation. In the long-term, our research program will facilitate new opportunities for RNA-based diagnostics and therapies that will be applicable to a wide array of human diseases.